Adeno-associated virus (AAV), a member of the Parvovirus family, is a small nonenveloped, icosahedral virus with single-stranded linear DNA genomes of 4.7 to 6 kb (Mr. 1.5–2.0×106). AAV is assigned to the genus, Dependovirus, because the virus was discovered as a contaminant in purified adenovirus stocks. AAV's life cycle includes a latent phase at which AAV genomes, after infection, are site specifically integrated into host chromosomes and a lytic or production phase in which, following either adenovirus or herpes simplex virus super-infection, the integrated genomes are subsequently rescued, replicated, and packaged into infectious viruses. The properties of simple genomic structure, non-pathogenicity, broad host range of infectivity, including non-dividing cells, and potential site-specific chromosomal integration make AAV an attractive tool for gene transfer. Recent studies suggest that AAV vectors may be the preferred vehicle for achieving stable gene expression.
To date, six different serotypes of AAV (AAV1–6) have been isolated from human or non-human primates (NHP), well characterized and vectored for gene transfer applications. All of them have been isolated as infectious viruses from either contaminated adenovirus preparations or tissues specimen of primate and non-human primate origin. Among them, AAV1 and AAV4 were isolated from non-human primates; AAV2, 3 and 5 were derived from humans, and AAV6 was a contaminant of a human adenovirus preparation.
Recently, taking advantage of the AAV's ability to penetrate the nucleus, to integrate into host and establish a latent infection in the absence of a helper virus co-infection, we invented a polymerase chain reaction (PCR)-based strategy for isolation of sequences of novel AAVs from cellular DNAs prepared from different tissues of non-human primate origin. Using this strategy, we have isolated at least 16 molecular types and 8 molecular subtypes of novel AAVs, generated recombinant viruses for two of them to evaluate their performance in gene transfer applications.
There remains a need in the art for reliable methods of identifying and isolating AAV virions from cellular sources.